Whole blood separation method and test using same

ABSTRACT

A METHOD FOR SEPARATING WHOLE BLOOD INTO A SUBSTANTIALLY COLORLESS FLUID AND THE RED CELL COMPONENTS OR RESIDUE COMPRISING CONTACTING THE WHOLE BLOOD WITH A WATERSOLUBLE SALT HAVING AN INORGANIC CATION SUCH AS POTASSIUM CITRATE, AMMONIUM SULFATE, ZINC SULFATE AND THE LIKE. IN A PREFERABLE USE OF THIS MEANS TO EFFECT A TEST DEVICE AND METHOD FOR DETECHING SOLUBLE CONSTITUENTS IN WHOLE BLOOD, A MATRIX CONTAINING THE SALT IS POSITIONED ADJACENT TO A TEST REAGENT SPECIFICALLY REACTABLE WITH AND GIVING A DETECTABLE RESPONSE TO SAID SOLUBLE CONSTITUENT, THE WHOLE BLOOD FIRST BEING CONTACTED WITH THE SALT AND THE COLORLESS FLUID THUS OBTAINED THEN CONTACTED WITH THE TEST REAGENT.

WHOLE BLOOD SEPARATION METHOD AND TEST USING SAME Filed July 13, 1967IIZI DIRECTION OF ZFLUID FLOw I II I IIIIII.

DIRECTION OF I FLUID FLOW FIGURE 3. FIGURE 4.

FIE'F I m I FIGURE I. FIGURE 2.

DIRECTION OF FLUID FLOW In-Z FIGURE 6. DIRECTION OF FLUID FLOW fDIRECTION OF FIGURE FLuID FLOw I FIGURE 5.

INVENTOR MARI ON C FETTER BY\A7\I FIGURE 8. ATTORNEY United StatesPatent U.S. Cl. 23-230 3 Claims ABSTRACT OF THE DISCLOSURE A method forseparating whole blood into a substantially colorless fluid and the redcell components or residue comprising contacting the whole blood with awatersoluble salt having an inorganic cation such as potassium citrate,ammonium sulfate, zinc sulfate and the like. In a preferable use of thismeans to effect a test device and method for detecting solubleconstituents in whole blood, a matrix containing the salt is positionedadjacent to a test reagent specifically reactable with and giving adetectable response to said soluble constituent, the whole blood firstbeing contacted with the salt and the colorless fluid thus obtained thencontacted with the test reagent.

Recently numerous simple test devices have been developed for the rapidanalysis of body fluids for various constituents which, if found to bepresent in excess of a predetermined amount, indicate the probability ofa pathological condition. Such tests include, inter alia, means fordetecting glucose and other sugars such as galactose in urine and blood,protein in urine, ketone bodies, urea nitrogen, uric acid, phenylalanineand certain enzymes, only to mention a few. Of the many tests andmethods devised, the detection of various soluble constituents in wholeblood has been found to be particularly difficult. The reason for thisdifficulty resides in the fact that the known rapid and simple testdevices almost invariably utilize chromogenic or other visual responsesto indicate the presence or absence of the constituents being detectedand the presence of the red coloration in whole blood usually seriouslyinterferes with this analysis. Of the other simplified test proceduresand compositions which employ responses other than chromogenic, thepresence of whole blood likewise seriously interferes with theobservation or measurement of the positive response. These other methodsdepend upon such chemical phenomena as precipitation and enzymecatalyzed reactions, gas formation, coagulation, agglutination,formation of ultraviolet and infra-red absorbing molecules andfluorescence, only to mention a few.

Various means have been proposed for separating and remow'ng the highlycolored red cell components from whole blood prior to, for example, theanalysis thereof. One of the simpler of these methods involves the useof a bibulous carrier member which is impregnated with a test reagentcomposition and coated with a semi-permeable membrane which effectivelyacts as a means for screening out large molecules such as hemoglobin,but permits the passage of smaller molecules and ions in solution. Aclear fluid containing the constituent being sought then enters the testreagent portion of the carrier causing a chromogenic reaction in thecase of a positive test. The

ice

strip is then rinsed with water to remove the excess blood andhemoglobin from the surface of the carrier.

This method, however, is rather cumbersome and laborious and requires anextra manipulative step, i.e., rinsing with water. In mass screeningprograms this extra step can amount to a considerable loss in time andefficiency. Moreover, the membrane can screen out larger molecules insolution which would preclude these molecules from reaching the testreagent and hence render the method inoperative for such determinations.

It is therefore an object of the present invention to provide a simplemeans for separating the highly colored red cell components or redcoloration from whole blood. It is another object of the presentinvention to provide a simple and rapid test device for detectingcertain soluble constituents in whole blood. It is still another objectto provide a unitary test device whereby whole blood may be analyzed forcertain soluble constituents using a single manipulative step. It is afurther object to provide a means for analyzing blood for solublemolecular constituents which would be removed by a membrane separatingmeans.

It has now been found that certain chemical compounds are effective incausing a separation of the highly colored red cell components of wholeblood from the fluid in which these red cell components are found. Theexact chem ical reaction involved in this phenomonon is not yet known.The result, however, is a highly effective means for removing thiscolored component from the whole blood, thus allowing a faciledetermination of constituents in the remaining fluid.

Specifically, it has been found that if whole blood is caused to contactcertain chemical compounds, the resulting blood fluid after separationfrom the residue is usually slightly straw colored or is imbued withonly a slight color. This slightly colored fluid may then be contactedwith a test reagent which specifically reacts with a particularconstituent to be detected in the blood fluid. Specific bloodconstituents which have been found amenable to the concepts disclosed inthe present invention, include, inter alia, glucose, galactose, urea,uric acid, phenylalanine and various enzymes.

As used hereinafter, the following terms are defined as follows:detection is defined as the quantitative determination of the particularconstituent being analyzed as well as the qualitative detection thereof;separating reagent is defined as a chemical or mixture of chemicalswhich effect a separation of the highly colored red cell components ofblood from the remaining fluid; test reagent is defined as a chemical ormixture of chemicals which cause an observable or detectable reactionwhen contacted with the substance being detected; matrix is defined asany physical means whereby the test reagent and/or separating reagent isconfined and positioned so that the blood may make contact therewith;whole blood is defined as blood containing all the originally present invivo constituents and may include anti-coagulants and other adjuvants.

Although the separation means of the present invention has utility inmany areas of blood technology as noted hereinabove, it may be andpreferably is utilized in conjunction with a test composition which isspecifically reactable with a soluble constituent contained in the bloodfluid and an analysis or estimation of this soluble constituent isdesired. In such a system, the whole blood may simply be initiallycontacted with the separative reagent and then, after removal of theresidue, the remaining fluid contacted with the test reagent. However,the test reagent and separating reagent are preferably incorporated witha matrix to effect a single entity test device. Since the function ofthe matrix is merely to suspend and contain the reagents, whether thefunction of the reagent be to separate the red coloration from the bloodsample or to detect the constituent being sought, the composition orphysical makeup of the matrix may comprise many forms, such as cellulosefibers as found in paper, synthetic fibers, food sticks, cloth, spongematerials, argilaceous substances, conduits, and so forth. The onlyrequirement of the matrix is that it suspend and contain the reagents.However, in utilizing the separating reagent, it is necessary that thematrix allow the colorless or only slightly colored resultant fluid topass through or at least to flow therefrom in order that it may contactthe test region for analysis. The preferable matrix material of thepresent invention is bibulous filter paper.

The test system of the present invention may comprise a single matrixwhich contains both the separating reagent or reagents and the testreagent in such a way that the Whole blood first contacts the separatingreagent and the substantially colorless fluid thereby obtained thencontacts the test reagent. In employing the single matrix test systemthe separating reagent must be compatible with the test reagent, bothfrom a reaction standpoint, and from a chemical stability standpoint,and the matrix must be of such a design that when the blood samplereaches the area of the device where the positive or negative responseis read, the fluid must be substantially free of the red coloration. Insuch an embodiment, a single bibulous paper strip is first impregnatedthrough and through with the test reagent and subsequently the surfaceof the matrix coated or impregnated with the separating reagent. In sucha test device the whole blood is first contacted with the separatingreagent and the test response observed in an area not initiallycontacted with the blood and to which the substantially colorless fluidhas migrated. For example, blood is applied to a side of bibulous paperstrip which is coated with the separating reagent and the responseobserved on the reverse side.

Although a single matrix test device may be constructed as indicatedhereinabove, test systems of the present invention are preferablyfabricated so that the separating reagent is contained in or on a firstmatrix and the test reagent in or on a second matrix. In so preparingthe test device, there is no danger from chemical incompatibility orinstability and only a minimal amount of separating reagent is carriedinto the test reagent area of the test device.

In regard to the positioning of the matrices when a dual matrix systemis utilized, it is to be noted that the test device utilizing theseseparating and analyzing means must be positioned so that whole bloodmust initially contact the first matrix containing the separatingreagent and the fluid must flow through or from this first matrix to thesecond matrix containing the test reagent. In the simplest and preferredform of the present invention, the matrix material is cellulose filterpaper which is impregnated in separate areas thereof with the separatingreagent and test reagent. This system may involve a strip of cellulosepaper, an end portion of which is impregnated with the separatingreagent and immediately inwardly of said impregnated end portion anadjacent or contiguous area of said strip is impregnated with the testreagent. It should be noted that although the term dual matrix is used,the matrix itself may be an integral unit.

Another embodiment of the test device of the present invention involvesthe positioning of the first and second,

matrices in laminate relationship, i.e., the first matrix comprising astrip of filter paper impregnated with the separating reagent isoverlayed or superimposed on a second strip of filter paper impregnatedwith the test reagent.

Specifically, the positioning of the various matrices and reagents maybe better understood by reference to the drawings wherein:

FIG. 1 shows a side view of a strip 3 of carrier material forming acontinuous matrix, a portion 1 near the end of which is impregnated withthe separating reagent and an adjacent portion 2 inwardly thereof isimpregnated with test reagent. FIG. 2 is an edge view of this same teststrip.

In utilizing this device, the end portion of strip 3 containing thereagents is immersed in the blood to be analyzed to a depth preferablysufficient to cover the major portion of the area 1 but not to a depthto completely cover said area. The colored portions of the blood areretained in the area 1 of strip 3 and a substantially colorless fluidthen migrates along the strip into contact with the test reagent in area2, giving a detectable response if the constituent being tested for ispresent in the blood fluid.

FIG. 3 is a side view of a strip 4 of transparent or translucentmaterial having directly afiixed thereto a matrix 5 shown in dottedlines and impregnated with a test reagent. FIG. 4 is an edge view ofthis same test strip. Immediately overlaying the matrix 5 is a secondmatrix 6 impregnated with the separating reagent.

In using this device, blood to be tested is applied to the surface ofthe separating reagent matrix 6, and the colored portions of the bloodare retained in said matrix 6, where as the colorless portion thereofmigrates through to the test reagent matrix 5. The color response ifany, in matrix 5 is observed through the transparent or translucentmaterial 4.

FIG. 5 shows a conduit of transparent or translucent material, forexample of plastic, which forms an outer matrix within which a testreagent 8 and separating reagent 9 are positioned either with or withoutan additional internal matrix. In use of this device, blood is pumped orallowed to gravity flow through the device from the separating reagent 9which retains the colored portions thereof, to the test reagent 8. Thecolor response if any, is detected in the test reagent area 8.

FIG. 6 shows a strip of carrier material or first matrix 10 into or uponwhich a test reagent is incorporated. Discs of a matrix 11 into whichthe separating reagent is impregnated are laid upon the matrix 10containing the test reagent. In use of this device blood to be tested isapplied directly to a matrix 11. The colored portions of the blood areretained in the matrix 11 and the colorless portion thereof flows intothe matrix 10. Removal of the matrix 11 to which blood was applied,permits observation of any color reaction occurring in the matrix 10containing the test reagent.

FIG. 7 shows a form of the invention similar to that shown in FIG. 6 butused differently. In FIG. 7 a separating reagent and a test reagent areseparately impregnated into bibulous members 12 and 13, and such membersjoined together in laminate relation as shown. A drop of blood, notshown, is placed upon a suitable surface and the laminate unit is thenplaced upon the blood drop with separating matrix 12 down. The coloredportions of the blood drop are retained in matrix 12, and the clearfluid flows upward and into the test reagent matrix 13 to cause adetectable color response in the latter matrix in the case of a positivetest.

FIG. 8 shows a circular planar bibulous matrix member 14 having acentral portion 15 thereof impregnated with a separating reagent. Anannular portion 16 of the member 14 surrounding the portion 15 isimpregnated with a test reagent. Blood to be tested is applied to thecentral portion 15 of the device. The colored portions of the blood areretained in the area 15 and the colorless portions thereof migrateradially outward into the test reagent area 16 to cause a detectablecolor response therein in the case of a positive test.

Referring now to the separating reagent which effects the removal of the'highly colored red cell components from full blood, it has been foundthat water-soluble salts, inorganic with respect to the cation, invarying degrees, effect such a separation. Before these salts arediscussed, however, it should be stated that the chemical and/orphysical action of such salts is not completely understood and thatcertain salts appear to act in a different manner and effect a differenttype of separation. In order to appreciate these differences, it shouldbe noted that red blood cells or erythrocytes are complex entities whichare normally suspended intact in the fluid phase of blood. When bloodnormally clots or coagulates, the red cells congregate or clump andleave behind a straw colored rather viscous fluid. However certainchemicals rupture the red cell and release the constituents therein,including the highly red colored constituent called hemoglobin or thespecific constituent of hemoglobin called heme. This rupturing of thered cell is known technically as hemolysis.

In the present invention, certain of the salts have been found to effecta rather complete separation of the red cell components from whole bloodleaving behind a clear colorless or straw colored fluid. In this case,obviously, the entire red cell has been removed. Other salts effect whatappears to be a type of hemolysis, and since it is not known withcertainty what the exact chemical mechanism is in this case, it ispreferable to call this phenomonon blood staining. In this situation itwould appear that the red cell is ruptured and releases a soluble redcoloration which may or may not be completely removed by the chemical inuse. However in certain instances, this hemolysis or blood staining maybe desirable since analysis may then be performed on the red cells orcomponents therein.

Moreover, it has been found that the salts of the present invention areeffective in removing the red cell constitu ents from whole blood eventhough anti-coagulants have been added to the blood. Specifically thewhole blood may contain anti-coagulants such as heparin orethylenediamine tetraacetic acid (EDTA) and yet the anti-coagulatedwhole blood remains amenable to the separation means disclosed herein.

The salts found effective as separating reagents in the presentinvention are water-soluble salts which are inorganic with respect tothe cation. From a practical standpoint, the salt must be non-volatileand should not decompose to any appreciable extent under the conditionsof preparing and utilizing the test device. Moreover, since at least aportion of the salt is solubilized and is carried into the test reagentpart of the device, the salt must be compatible with the test reagent.However, the situation where the salt is carried into the test reagentmay be advantageously utilized where the salt becomes an integral partof the test reagent system. This concept will be more fully elucidatedhereinafter. For the purpose of the present invention, the termwater-soluble salt is defined as a salt having a solubility in distilledwater, without the aid of solubilizers, of at leastabout 1 gram perliter at 20 C. Among the many salts which are operable in the presenttest device which act as a separating reagent are: ammonium acetate,ammonium bromide, ammonium chloride, ammonium citrate, ammonium formate,ammonium nitrate, ammonium sulfate, ammonium tartrate, lithium sulfate,nickel(ous) sulfate, potassium bromide, potassium chloride, potassiumcitrate, potassium nitrate, potassium oxalate, sodium bromide, sodiumchloride, sodium citrate, sodium fluoride, sodium lactate, sodiumnitrate, sodium phosphates, sodium sulfate, barium acetate, calciumchloride, calcium iodide, ferric chloride, magnesium sulfate, zincsulfate, ferric ammonium sulfate, sodium borate, sodium molybdate,ammonium lactate and ammonium succinate.

In preparing the test devices of the present invention and particularlyin regard to the separating reagent, the amount of reagent used willusually depend upon the particular chemical selected to perform thefunction of removing the red coloration from the whole blood. Since itis diflicult to assess the amount of a particular salt which isimpregnated into a carrier member, this facet of the present disclosurewill be defined in terms of the concentration of separating reagent inthe solution being used to impregnate the carrier member. Solutions ofsalts having a concentration of about from 1% to about 60% by weight maybe used. However for reasons of economy and efliciency, a concentrationof about 5% to 10% is preferable. Solvents other than water may be useddepending on the separating reagent solubility characteristics. Afterthe carrier member has been contacted with the separating reagent it iseither allowed to dry at room temperature or dried at elevatedtemperatures. Obviously, if the reagent is heat labile, lowertemperatures must be employed. Moreover, if lower concentrations ofseparating reagent are employed when a single matrix is used, it may benecessary to increase the distance through which the blood passes, e.g.,by increasing the area of the matrix impregnated with the separatingreagent in order to effect a complete separation of the red cellcomponents from the whole blood.

When a two matrix form of the present invention is prepared, it is theusual practice to separately impregnate the matrices for the separatingand test reagents and then to physically joint the impregnated matricesin laminate relation. In the case, however, where separate areas of thesame carrier are utilized these separate areas may be individually orsequentially impregnated depending on the apparatus used to produce thetest device.

In one embodiment of this invention (not shown) the same matrix is usedto retain the separating reagent and the test reagent in admixture. Toproduce this embodiment a single impregnation of a solution containingboth reagents or sequential impregnation of the same area with separatesolutions of the reagents may be employed. This situation, of course,likewise depends upon the solubility characteristics of the test reagentused. In practice, the sequential impregnation technique necessitatesthe drying of the test device to remove the solvent before the nextimpregnation is utilized.

As noted hereinabove, the preferable matrix material of the presentinvention is bibulous filter paper. Such paper has been found to beextremely satisfactory for use as a matrix material for suspending andpositioning both the separating reagent and the test reagent. Filterpaper has been found to have particular utility in retaining theseparating reagent, and in this regard the type of filter paper used hasbeen found to influence the effectiveness of the separation process. Asknown to those skilled in the art of basic chemistry, filter paper canbe obtained in a variety of thicknesses and porosities. Since use of thedevice requires the separating reagent to at least momentarily contactthe whole blood and the substantially colorless fluid must be allowed toemerge therefrom, both of the above factors influence the efficiency ofthe system. When coarse porosity paper is used, the thickness thereofmust be suflicient to allow the minimal contact time between the wholeblood and the separating reagent. The reverse of this, of course, islikewise true; and when a fine porosity paper is utilized, a relativelythin sheet may be employed. The proper balance between these factors, aswell as a judicious selection of the concentration of separating reagentin the impregnating solution is well within the experimental techniquesused by those skilled in the art of preparing test devices such as thosedescribed in the present specification.

An additional novel and advantageous feature of the present inventionresides in the fact that in cetrain instances the separating reagent maybe utilized as an integral part of the test reagent and thus perform adual function in the test system. For example, in an enzymatic glucosetest system which generally comprises glucose oxidase, a buffer, anoxidation-reduction indicator and peroxidase, a combination of potassiumiodide and sodium molybdate may be substituted for the peroxidase. Whenwhole blood is applied to a system utilizing this combinationmolybdate-iodide salt, the salts act as both the separating reagent andas the peroxidase substitute. When the potassium iodide-sodium molybdateimpregnating matrix is in contiguous relation to the test mersed intothe solutions, removed, allowed to drain and dried in an oven at 40 C. Apreliminary screening was carried out by placing drops of EDTA(ethylenediaminetetraacetic acid) anti-coagulated and heparinanticoagulated whole blood on separate impregnated filter reagent matrixand the blood is applied to the separating papers and observing the zoneimmediately beyond the matrix, the substantially colorless fluidemerging therearea of the drop of blood for a fluid which separates fromcarries the soluble salts into the test reagent matrix and migrates awayfrom the area of application. As and the test functions in the normalmanner. In such a shown in Table I, in some cases the separated zoneconsituation, the concentration of salt must be adjusted to tained aclear colorless fluid while in certain other meet the needs of both theseparating reagent and the test cases there was some degree of hemolysisor blood stainreagent. In a similar situation, when the test reagentconing. In the cases Where the salt caused blood staining, the tainssalts which are effective as separating reagents, then outer peripheryof the wicking fluid was relatively colorthe concentration of saltutilized primarily as a separating less. In some cases a double zoneformed. reagent may be correspondingly decreased. From the aminatedstrip test devices were then prepared as above, it is apparent that thepresent invention allows shown in FIGS. 3 and 4 of the accompanyingdrawings. for considerable latitude in selecting reagents and the con-The test reagent part 5 of the strip was impregnated with centration andplacement thereof. This manipulation of a glucose sensitive compositionprepared by first adding, the test device is well within the scope ofone skilled h agitation, grams of glucose Oxidase, 01012 in the art withthe present disclosure at his disposal. gram of per idase, 0.269 gram ofpotassium dihydro- The mode of use of the present invention varies withgen phosphate, 0.004 gram of sodium monohydrogen the particularembodiment employed. Generally, in utiphosphate and 10.0 ml. of acetoneto a small flask conlizing the single matrix test srtip system in whichthe retaining about 50 ml. of distilled water. The total volume agentsare present, a drop or two of blood is placed of the solution wasbrought to 100 ml. by the addition of on the face of the impregnatedmatrix held in a horizontal distilled water. A second solution was thenprepared by position and allowed to penetrate into the body of thedissolving 0.42 gram of ortho-tolidine in a small quantity test device.After the colorless fluid migrates down through of acetone and bringingthe total volume of the solution to the matrix, the device is turnedover and any color re- 100 ml. with acetone. Pieces of Schleicher andSchuell filsponse is observed on the side opposite the site of appliterpaper 'No. 2316 were then dipped into the first solucation. Ifpenetration is not complete, more blood is added tion, allowed to drainand dried at These Same to the previous site of application. This typeof device may pieces of filter paper were then dipped into the secondalso be partially immersed into the whole blood to b solution, allowedto drain and dried at room temperature. tested and withdrawn. Some ofthe blood fluid will mi- Upon drying, the impregnated paper was a lightyellow grate away from the immersed area and be decolorized by theseparating reagent. Any color response is ob- The paper impregnated withthe test reagent was then servedinthis area. cut into small squares 5about /2 cm. x /2 cm. and In utilizing the two matrix test device, thesame prosuitably aflixed to an end portion of the strip 4 of clearcedures as above apply except that the blood is always flexiblepolyvinyl chloride film about 8 cm. x /2 cm. The applied to the matrixcontaining the separating reagent papers impregnated with the separatingreagent as shown and any color response is observed in the matrix coninTable I were similarly cut into strips 6 about /2 taining the testreagent to which the colorless fluid has cm. x 1 cm. and placed directlyover the test reagent migrated. paper 5 previously aflixed to the clearstrips 4. The over- The mode of use and the method of preparing thepreslapping ends of the separating reagent paper 6 were also ent testdevice will be illustrated by the following exsuitably aflixed to theclear strip as shown in FIG. 2. The amples. These examples will alsoserve to illustrate the 40 test devices thus prepared make up thelaminated strips broad scope of the present invention as well as some ofthe used as indicated in Table I. specific concepts disclosed herein.The examples are not, The test devices were placed on a level surfacewith however, to be considered as placing any limitation on theseparating reagent impregnated paper facing upthe full scope of thepresent invention. ward. A drop of EDTA anti-coagulated whole bloodcontaining 150 mg. percent glucose was then placed directly EXAMPLES 135 in the center of the separating reagent paper 6. Two minutes wereallowed to elapse (except where noted in In the following examples,solutions of the salts listed Table I) and the strip 4 was turned overto expose the in Table I were prepared by dissolving the indicatedmotest reagent area 5 under the clear plastic strip. The lar quantity ofthe salt in distilled water. Pieces of two color reaction observed isnoted in the column designated types of filter paper as indicated inTable I were im- Reaction in Table I.

TABLE I S and S No. 2316, Separating S and S No. 5700, Separatingreagent matrix reagent matrix Preliminary Preliminary screenmg screeningExample Cone. of Separa- Blood Laminated strip Separa- Blood LaminatedSeparating reagent (salt) No. salt tion staining reaction tion stainingstrip reaction Ammonium acetate 1 1 Red blue Brown blue. Ammoniumbromide. 2 Green. Ammonium chloride. 3 L. green. Ammonium citrate.-. 4Green. Ammonium formats. 5 Red brown. Ammonium 11itrate 6 D.green D.green. Ammonium sulfate 7 Green :6} Green. Ammonium tartrate..- 8 .d0 L.green. Lithium sulfate 9 (L. green) 5533;; Nickel (011s) suliate 10 Graygreen- Blue black. Potassium bromide" 11 Blue green" Blue green.Potassium chloride. 12 Green Green. Potassium citrate 13 0 L- green. 56}M. green.

TABLE IContinued S and S No. 2316, Separating S and S No. 5700,Separating reagent matrix reagent matrix Preliminary Preliminaryscreening screening Example Cone. of Separa- Blood Laminated stripSepara Blood Laminated Separating reagent (salt) No. salt tion stainingreaction tion staining strip reaction Potassium nitrate.... 14 D. greenBrown green. Potassium oxalat 15 Green.. i reen. Sodium bromide- 16 dDo. Sodium chloride. 17 0- Sodium citrate... 18 0 Do. Sodium fluoride-19 L. green. Sodium lactate- 20 Blue brown Sodium nitrate 21 D. green.Sodium phosphate 22 i L. green. Sodium sulfate 23 (L. green) g Bariumacetate 24 Green Green. Calcium chloride 25 .....do (D Do.

. green. Calcium iodide 26 (131116 brown) L. green. Fem'c chloride... 27O 0 Magnesium sulfat 28 Green L. green. Zinc sulfate 29 (L. Green) 0Ferric ammonium sulfate"--. 30 Saturated--- 0 0 g Do 31 Slurry 0 0Sodium borate.-.. 32 L. green. Sodium molybdate- 33 o- Ammoniuni lactate34 Green. Ammonium succinate. 35 D. green Do.

1 .gmngionium acetate impregnated paper dried at room temperature (20C.) Z on e.

3 Combination of about 2 parts NazHPOi and 1 part NaHzPO-i.

4 Ferric chloride not compatible with glucose test system Abbreviationsand symbols used.Conc.=Concentration oi impregnating solution; D=Dark;L=Light; Mod.=Moderatc; Soln.=Solution; 0=negative result; +=positiveresult; -=test reagent area not wetted in 5 minutes; =resu1ts after 5minutes.

EXAMPLE 36 References Cited A separating reagent solution was preparedby adding UNITED STATES PATENTS 100 mg. of sodium chloride to 2 ml. of0.9% saline solu- 233L758 5/ 1942 Galat 252408 tiOIl. One drop of thissolution was placed in a spot plate 0 2,893,844 7/1959 (390k 252'408Xand one drop of blood added thereto. After mixing for 15 3,000,836 9/1961 Glnsbllrg 252-403 seconds with an applicator stick, an end portionof a strip 3,009,862 11/1961 Dobnck 25240874: of filter pap r was Placedin the mixture. A colorless fluid s :23- mi rated 1 h filt 1116 y 10 i 53.3 .53 aper 3,011,874 12/1961 Deutsch 23-23OX(B 1. A process fordetecting a soluble constituent in 3,016,292 1/1962 Baver et 10) wholeblood comprising contacting the whole blood with 3,048,475 8/1962 P 10)a first matrix having incorporated therewith the dried 3,069,330 12/1962Babsoll 195103-5 residue of a separating reagent solution of about from3,123,443 3/1964 Y 10 1% to 60% by weight of a water soluble,non-volatile salt, 3,235,337 2/1966 23253 inorganic with respect to thecation, allowing the substan- 3,266,868 8/1966 Harvlu 23253(TP) tiallycolorless fluid resulting from contacting the whole 3,341,427 9/1967Evans et 195-1O3-5 blood with said separating reagent to contact asecond 3,349,006 10/1967 Albaum 10) matrix containing a test reagentspecifically reactable with 3,359,072 12/1967 y et 1o) said constituent,said second matrix being in adjacent position to said first matrix, andobserving the reaction be- FOREIGN tween the constituent and the testreagent in an area of 1,037,155 7/ 1966 Great Brltalll 23253 the secondmatrix other than the point of contact.

2. A process as in claim 1 wherein the first matrix and MORRIS WOLKPnmary Exammer the second matrix are a bibulous paper material. RICHMAN,Assistant er 3. A process as in claim 1 wherein the separating reagentsolution comprises from about 5% to about 10% by w ight of said salt,23253; 103.5; 210-25; 252408

